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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: Impact of HIV-1 Vpr manipulation of the DNA repair enzyme UNG2 on B lymphocyte class switch recombination

Fig. 4

Impact of Vpr on CSR in immortalized B-cells. a Left panel, CH12F3 cells were transduced with VLP HA-Vpr at a M.O.I. of 10. 48 h later, UNG2 content was assessed by western blotting with anti-UNG2 antibodies as described in Materials and methods. Lanes were cropped from blot membranes serially probed with anti-UNG2 then with anti-αtubulin antibodies (see Additional file 2: Supplementary dataset 2). Right panel, CH12F3 cells transduced with VLP HA-Vpr (M.O.I. of 5) were examined for UNG activity at days 0, 1, 2, 3, 4 and 7. b After 24 h in culture CSR was induced by IL-4/α-CD40/TGF-β stimulation for 3 days. Class switch efficiency from IgM-to-IgA isotype was evaluated by flow cytometry by measuring the  % IgGA+ B220+ cells (left panel). DNA from corresponding cells was extracted and uracil content (dUrd per 105 bp) was determined by LC/MS/MS. Cells transduced with VLP ΔVpr and mock-transduced cells are shown as controls. c CH12F3 B-cells were transduced with increasing MOI of VLP HA-Vpr. 24 h later, corresponding isotype switching was induced by cytokine stimulation for 3 days and evaluated by flow cytometry. Values are the mean of duplicate experiments ± SD. Corresponding UNG activities were measured in the presence or absence of the UNG inhibitor UGI. Stim: stimulated; Unstim: unstimulated

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